PROJECT SUMMARY Despite great advances in genome editing technology, few clinical gene delivery therapeutics currently exist. Concerns over unwanted genome alterations that can be caused by current technologies have tempered excitement and delayed translational progress. New tools are needed to allow researchers and clinicians to direct DNA to a single sequence without concern for off-target disruption. Safer technologies could broaden genomic therapy beyond terminal disorders and warrant the treatment of increasing numbers of candidate diseases. Unlike current gene targeting approaches that act passively, recombinase proteins are capable of actively inserting DNA into the genome. Several recombinases are highly efficient at genomic integration, although no recombinase is single-site specific. In this proposal, the exquisite site specificity of the CRISPR Cas9 system will be leveraged with the robust efficiency of actively integrating recombinases. Directed evolution is capable of imparting entirely new activities on proteins. New evolution technologies are orders of magnitude faster at improving proteins and could be used simultaneously to increase both DNA binding specificity and DNA integration efficiency. There exists a need for safer technologies capable of efficiently delivering therapeutic DNA to desired sequences in the genome. The long-term goal is to generate a targeting technology capable of efficiently inserting DNA at a single location in the genome without unwanted off-target alterations. The overall objective of this application is to demonstrate the proof-of-concept that rational design and directed evolution are capable of improving this specificity. The central hypothesis is that modifications to a recombinase will improve the targeting specificity to sequences in the human genome. This hypothesis has been formulated based on literature reports and preliminary experiments from the applicant's laboratories demonstrating that fusion proteins consisting of custom DNA-binding proteins fused to the piggyBac transposase are capable of targeting desired locations in the genome. A key innovation in this proposal is the use of a rapid directed evolution technique called phage-assisted continuous evolution (PACE) that is capable of performing dozens of cycles of evolution in a single day. The central hypothesis will be tested by pursuing two specific aims: 1) Improve genome targeting specificity of recombinases using rational design; and 2) Improve integration efficiency and specificity of recombinases using directed evolution. This proposal is significant because it develops a new tool capable of safely and efficiently directing therapeutic genes to desired locations in the genome. This technology applies to both preclinical research using gene targeting as well as potential treatments of any disease requiring gene replacement. The proposed innovative modifications, including a novel directed evolution approach, have not been previously explored. long-term success would result in a novel technology capable of both specifically targeting the genome and actively integrating DNA; a critically needed tool that does not currently exist.